Search

Autoantibodies against insulin, C-peptide, and glucagon were determined by radio-binding assay in 63 new-onset Type 1 (insulin-dependent) diabetic patients as well as in 70 controls. Plasma peptide binding was determined by means of 125I-labeled peptides and charcoal-dextran separation technique. Binding values exceeding the mean plus three standard deviations of the controls were considered as antibody-positive. Sixteen patients (25%) were positive for IAA, as 6 (10%) were positive for CAA and 2 (3%) for GAA. Of all control subjects, none were positive for either IAA or CAA, whereas 2 (2%) had GAA. The mean 125I-glucagon binding in the patients' group was, however, slightly enhanced and could be suppressed to normal values by excess unlabeled glucagon. The presence of IAA and/or CAA was significantly associated with more severe symptoms at diabetes manifestation. These results indicate that in new-onset Type 1 diabetics autoimmunity arises against all the insular peptides tested but is predominantly directed against those antigens secreted from the beta cells. Nevertheless, extremely low-binding GAA seem to be common in these patients. The determination of IAA/CAA might be useful in detecting a possible heterogeneity of Type 1 diabetes with regard to its clinical mode of manifestation.

Neither injection of complete Freund's adjuvant (CFA) alone nor the administration of low doses of streptozotocin (STZ) to rats produced remarkable histopathological changes in the endocrine pancreas, but treatment with the combination of both resulted in necrosis of beta cells. When the combination of CFA/STZ was given two times, necrosis progressed, and the beta cell reserve was depleted to such an extend that persistent hyperglycemia ensued. These changes were associated with a significant reduction in the apparent islet size. A single injection of CFA induced pancreatitis and inflammatory lesions in the exocrine parenchyma with no insular involvement. Three injections caused extensive destruction of pancreatic acinar tissue but only moderate beta cell injury in the minority of islets. Apart from mild degranulation of beta cells, treatment with STZ did not produce histopathological changes in the pancreas. These results suggest that the acute inflammatory process induced by CFA may initially damage the beta cells, increasing thereby their susceptibility to the action of STZ.

To investigate whether the unexpectedly high C-peptide levels in some insulin-dependent diabetic (IDDM) patients are due to co-determination of proinsulin bound to circulating insulin antibodies, 36 randomly selected sera from IDDM patients were assayed for C-peptide immunoreactivity (CPR) after polyethylene glycol (PEG) extraction, preceding incubation with proinsulin binding antibodies (LAB + PEG) or without pretreatment of the sera. Recovery of proinsulin was checked by addition of 1 nmol/l proinsulin to all sera. Recovery was found to be 101.5 +/- 4.0%. The mean values of concentrations were significantly lower (p less than 0.001) after treatment with PEG and IAB + PEG compared to the untreated sera. There was also a significant difference (p less than 0.05) between sera extracted with PEG alone or after IAB + PEG-treatment. However, no correlation (p greater than 0.1) was found to bound insulin (total minus free insulin) or to insulin binding capacity (IBC) of the sera. If an antiserum is not available with very low cross-reactivity with proinsulin to determine human C-peptide then sera should not be extracted with PEG alone but after additional incubation with a proinsulin binding antiserum. In spite of the extraction in some cases unexplicably high C-peptide levels may still be expected.

Following optimization of the reaction conditions, e.g. concentration of oxidizing agents, reaction time, volume of reaction mixture, and pH, chloramine T and the new iodination reagent, Iodogen, were compared for their effectiveness in radioiodination of insulin, glucagon, human growth hormone (hGH), and rabbit anti-mouse IgG. The radioactive peptide hormones prepared were analyzed for the presence of aggregate and breakdown products by polyacrylamide gel electrophoresis (PAGE) at pH 8.9, the rabbit anti-mouse IgG was tested for the presence of low molecular weight damage products by gel filtration on Sephadex G-50. The results demonstrate that with respect to iodine incorporation, specific activity, and immunological reactivity either method can be used to prepare under carefully controlled conditions a wide range of tracers with high specific activity at minimal oxidation damage. These tracers are shown to be highly suitable in radioimmunoassays after previous purification by PAGE and gel filtration, respectively.

In the present study we characterized and compared the different molecular forms of glucagon-like immunoreactivity in extracts of peripheral plasma and hepatic metastases of a patient with pancreatic alpha-cell tumor. Plasma and tissue extracts were chromatographed on Sephadex G-50 columns. Immunoreactivity in the eluting fractions was assayed with an anti-glucagon antiserum that specifically recognizes the C-terminal region of the pancreas glucagon molecule. Total plasma glucagon-like immunoreactivity prior to surgery was 26.64 nmol/l and consisted of four peaks of immunoreactivity of apparent 9,000 mol wt, 5,800-5,400 mol wt, and 4,000 mol wt. Total glucagon-like immunoreactivity extracted from the hepatic metastasis was 47.41 nmol/g wet weight and eluted as two major peaks of immunoreactivity as follows: peak I, mol wt 3,800, corresponding to "true" 3,500 mol wt glucagon; peak II, mol wt 1,400, probably consisted of glucagon degradation products. The results clearly demonstrated that both plasma and glucagon-like immunoreactivity extracted from hepatic metastases were heterogeneous and comprised species corresponding not only to "true" glucagon but also to higher mol wt forms. The lack of higher mol wt forms of immunoreactivity in the hepatic metastases of the alpha-cell tumor suggests that this metastatic tumor tissue may contain an enzyme capable of converting the higher mol wt forms to smaller glucagon-like components whereas this degradative system seems to be defective in the primary tumor.

The age-dependent relationship of fasting immunomeasurable C-peptide to fasting immunomeasurable insulin (IRI) and IRI response to glucose was studied in 113 non-obese healthy subjects with normal glucose tolerance (oGTT according to the new WHO recommendations), ranging in age from 6 to 44 years. Fasting C-peptide concentration increased significantly in adolescents and adults when compared with children. The higher fasting C-peptide concentration in the adult group might be explained by the concomitant higher fasting blood glucose concentration whereas such relationship was lacking in adolescents. In contrast to this we failed to demonstrate such relationship with regard to fasting IRI levels. There was, however, a relationship between advancing age and early (IRI area 0-30 min), late (IRI area 30-120 min) and total (IRI area 0-120 min) insulin response to glucose. There was a significant correlation between fasting C-peptide concentration and estimates of IRI response in children and adolescents, whereas such relationship was lacking in adults. Based on these results, the present study demonstrates an age-related increase of the pancreatic beta-cell function which might be partly explained by the concomitant higher blood glucose concentration.

A sensitive radioimmunoassay (RIA) for canine C-peptide (CCP) was established using synthetic CCP, a specific antiserum, and rabbit anti-guinea pig serum. Radioiodination was performed according to a modified chloramine-T method. Tracer preparations were used for long as 6 weeks after iodination. The standard curve ranges from 0.028 to 3.0 nmol/l. The intra-assay coefficient of variation (CV) was 3-5% and the inter-assay CV was 6-9% in the optimal range between 0.3 and 0.8 nmol/l. The average recovery of CCP added to plasma samples was 100.6% (n = 9). Canine insulin, porcine proinsulin, bovine proinsulin, and human C-peptide exhibited no cross-reactivity. The mean fasting plasma CCP concentration was 0.089 +/- 0.021 nmol/l in normal dogs and -0.005 +/- 0.007 nmol/l (mean +/- SEM) in diabetic dogs, respectively.

In order to assess the validity of WHO criteria for the discrimination between Type I and Type II diabetes a cross-sectiona clinical study was performed in 84 normweight newly diagnosed diabetics with a mean age of 22 years. Taking into consideration clinical and biochemical characteristics of the carbohydrate and fat metabolism, the therapeutic requirement to maintain euglycemic metabolic control, the residual beta-cell function, the HLA phenotype and islet cell antibodies (ICA, ICSA) it could be shown that none of the tested criteria has the ability to distinguish between the types with absolute certainty. As shown by the frequency of the different markers in relation to the therapeutic requirements for euglycemic metabolic control as well as by the correlation analysis between the variables the discriminating validity of the markers decreased in the following sequence: diabetes associated HLA phenotype, residual beta-cell function, proneness to ketosis, age at onset, relative body weight. Neither the characteristics of the carbohydrate and fat metabolism nor the presence of islet cell antibodies contributed much to the differentiation between insulin-dependent and noninsulin-dependent diabetes.

The non-specific activation of the immune system by administration of complete Freund's adjuvant (CFA) was examined in two congenic Lewis rat strains LEW. 1A (RT1a) and LEW. 1W (RT1u) as a possible mean of amplification of the specific immune response, directed to pancreatic beta cells induced by multiple non-diabetogenic injections of streptozotocin (STZ). Rats were given intraperitoneally 0.5 ml CFA and 1 day later 25 mg/kg body weight STZ. This combined treatment was repeated twice at weekly intervals. Control groups received vehicle, STZ or CFA only with the same doses and at the same times. Only CFA/STZ-treated rats developed a persisting hyperglycaemia (greater than 15 mmol/l glucose) namely 3/18 (17%) LEW. 1W and 47/76 (62%) LEW. 1A rats. The pancreatic insulin content in these hyperglycaemic rats was reduced by 96.6% in LEW. 1A rats and by 93% in LEW. 1W rats measured 8 weeks after the last CFA/STZ treatment. The response to CFA indicated by an increase of number of peripheral leucocytes and relative spleen weight gain at 7 days after CFA administration, was higher in LEW. 1A rats compared with those of LEW. 1W rats. Spleen cells harvested 72 h/48 h after the first and second CFA/STZ administration showed a cytotoxic reaction to isolated syngeneic islets as measured by 51Cr-release in vitro. Control rats receiving vehicle, STZ or CFA only showed no cellular anti-islet cytotoxicity. The anti-islet cytotoxicity of spleen cells was only transient and disappeared after the third CFA/STZ administration. Anti-islet cytotoxic antibodies were not detectable in this short-term study.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of islet cell cytoplasmic antibodies (ICA) and islet cell surface antibodies (ICSA) at the time of diagnosis of type 1 (insulin-dependent) diabetes mellitus has been taken as evidence that autoimmune mechanisms are involved in the pathogenesis of the disease. The demonstration that ICSA in the presence of complement are preferentially lytic for beta-cells may be important in defining the role of these autoantibodies in the pathogenesis of type 1 diabetes. Because of the polyclonality of the immune response, the ICA and ICSA molecules of diabetic patient vary enormously in their binding parameters. For this reason we have generated monoclonal antibodies (MC-Ab) to islet cell antigens. In this study we investigate the effect of the two MC-Ab K28 A1 and K28 D6 resulted from the same fusion of the P3-X63-Ag8 murine myeloma cell line with the spleen cells of a Balb/c mouse immunized with rat islet cells on the hormone release of isolated rat islet in co-culture with the antibody-secreting hybridomas. The MC-Ab K28 D6 binds to both islet cell cytoplasmic and surface antigens, the K28 A1 is only reactive with cytoplasmic antigens. Surprisingly, in contrast to the monoclonal antibody K28 A1, K28 D6 enhanced the glucagon content and diminished the insulin secretion of the islets. Either the K28 D6 is directed to an epitope occurring on the beta- as well as alpha-cells or the antibody-mediated inhibition of the glucagon release results in a significantly reduced insulin secretion.

Using the micro-scale modification of a newly developed RIA kit for insulin, we established methods for the determination of free and total insulin in serum of insulin-treated diabetics. Precipitation with polyethylene glycol 6000 or acid alcohol extraction of sera was carried out to remove or to dissociate antibody-bound insulin. Both assays permit precise and accurate measurement of either serum insulin fraction. In 50 diabetic sera with tracer insulin binding of 0-97%, free (after equilibration of the sera at 37 degrees C) and total insulin levels as well as insulin antibody binding parameters were determined. There was a good correlation of free to total insulin levels with maximally 10-fold higher values of total insulin. Both free and total insulin were found to be correlated with the ability of the serum to bind insulin. In detail, binding affinities (i.e. the reciprocal of equilibrium dissociation constants) and binding site concentrations were evaluated which were shown to be positively correlated with free and total insulin levels as well. From these data we conclude that insulin antibodies in the serum may accumulate therapeutic insulin and function as a depot for delivering insulin in insulinopenic episodes (Keilacker et al., 1982 and 1986).

The ability of islets of Langerhans to release insulin (IRI) and glucagon (IRG) after stimulation with glucose and arginine was analyzed by using isolated perfused pancreas of Lewis-rats and by using perfused liver three months after syngenic portal islet transplantation. Transplanted islets preserve their functional integrity so that the shape and magnitude of IRI and IRG release, respectively, can be compared with normal islet reactivity. They are able to release insulin after stimulation with 16 mM glucose having a typical biphasic secretion profile. The islet and B-cell volume, as well as the insulin and glucagon content of the recipient pancreas, are decreased significantly three months after islet transplantation when compared with healthy controls.

Since mumps virus seems to be one of the most likely candidates in viral etiology of insulin-dependent diabetes (IDDM) we studied the possible relationship of glucose tolerance (75 g oGTT), beta cell function, diabetes associated HLA antigens, haptoglobin phenotype, islet cell antibodies (ICA) and islet cell surface antibodies (ICSA) in 125 subjects with antecedent mumps infection. Impaired glucose tolerance (IGT) was diagnosed in 3.2% (n = 4) but onset of diabetes did not appear within 14 months after mumps infection. There was no relationship between glucose tolerance and complications of antecedent mumps infection (e.g. pancreatitis, meningitis, orchitis). The prevalence rate of ICA was 76%. ICSA were detectable in about 36% of children and 62% of the adults tested (p less than 0.01). There was no relationship between ICA/ICSA and diabetes-associated HLA antigens, haptoglobin phenotype or beta cell function (fasting C-peptide and insulin response to 75 g oGTT). However, adults with circulating ICA were characterized by a significantly lower insulin response to glucose. Fifty two "risk" subjects characterized by IGT, diabetes associated HLA antigen(s), ICA or ICSA either alone or combined were studied again 26 months after mumps infection. No symptomatic diabetes appeared and IGT was diagnosed in one case only. ICA and ICSA persisted in more than 50% of subjects in whom ICA or ICSA were present 14 months after mumps infection. Since the used immunological techniques do not clearly distinguish organ-specific from non-organ-specific antibodies the results must be interpreted with caution. To summarize, the preliminary results do not support a close temporal relationship between mumps infection and the onset of IDDM. The pathogenetic role of mumps virus and ICA/ICSA and their possible relation to a slow progressive beta cell destruction has still to be determined.

Two different antisera which specifically reacted with either the C- or N-terminal region of pancreas glucagon were employed to determine the concentration of glucagon and glucagon-like immunoreactivity in extracts from submaxillary glands of rats. The content of glucagon and glucagon-like immunoreactivity increased with age and was found to be higher in adult males than in females. Pancreas glucagon immunoreactivity constituted only a small proportion of the total glucagon-like immunoreactivity. The role that glucagon-like peptides present in the submaxillary gland of rats might play in the glucose homeostasis remains still unknown.

A radioimmunoassay for the detection of monoclonal islet cell antibodies was developed using rat insulinoma cells as antigen carriers and 125I-labeled affinity-chromatographically purified anti-mouse Ig antibodies for detecting cell-bound mouse Ig. Prior to the assay cells had been attached to glass tubes by poly-dimethyl-diallyl ammonium chloride thus allowing to perform the assay as easy as a solid-phase immunoassay. Incubation protocol and cell number were chosen to ensure a high sensitivity of the assay. Results compared well with immunofluorescence findings. Of seven monoclonal islet cell antibodies tested for crossreactivity only one was displaceable by islet cell surface antibodies from diabetic sera. This antibody was induced by immunization with human islets whereas all others were from mice which had been autoimmunized with streptozotocin and complete Freund's adjuvant.

Forty-three newly diagnosed diabetic patients from Ethiopia were studied for the frequency of islet cell surface antibodies and other clinical features which are relevant to the aetiopathogenesis and classification of diabetes mellitus. In preliminary investigations of a small number of controls and noninsulin-dependent diabetics we found, as expected, no circulating antibodies. Four first-degree relatives of ICSA-positive sibs were negative, too. However, 3 out of 7 known insulin-treated diabetics displayed ICSA in the blood serum. In our study of newly diagnosed diabetics we found ICSA in 39% (17/43). Five patients who were assigned to the NIDDM subclass had no antibodies. 37 diabetics required insulin treatment after clinical diagnosis and 16 (43%) of these were ICSA-positive. There were no differences in the assessed clinical parameters between ICSA-positive and -negative patients. One ICSA-positive patient initially controlled with oral hypoglycaemic agents became insulin-dependent within our study period. Our observations in newly diagnosed diabetics from Ethiopia revealed a lower frequency of ICSA than in Caucasians, however, the results provide evidence for the occurrence of auto-immune phenomena in the aetiopathogenesis of diabetes mellitus in this ethnic group.

In a hospital-based study in northwestern Ethiopia some clinical and biochemical features of diabetes mellitus have been assessed to contribute to the problem of classification of diabetes in a tropical country. Diabetes requiring primary insulin treatment is presented by unequivocally elevated blood glucose levels and the classic symptoms of the disease. Newly discovered cases and readmitted rural diabetics show significantly lower body mass indices and 31% have been classified as underweight. The overall frequency of ketonuria at (re)admission was 45% together with moderately elevated or high 3-hydroxybutyrate serum concentrations. The hormonal status is characterized by a reduced beta-cell function. Serum concentrations of all carnitine fractions are lower in both normal and diabetic Ethiopians when compared with Caucasoids. Carnitine precursor amino acids are normal and the complete amino acid spectrum reveales no clear-cut pattern related to protein-energy malnutrition.

Injection of complete Freund's adjuvant (CFA) 24 h prior to treatment of rats with non-diabetogenic doses of streptozotocin (STZ) produced a reduction in pancreatic insulin content associated with ultrastructural changes and a decrease of the volume density of insulin-immunoreactive cells without affecting other islet cells. After one injection of CFA and STZ, pancreatic insulin content was decreased by 69% (p less than 0.01) within 48-96 h, while plasma glucose concentrations were not changed. The volume density of beta cells in pancreata of these rats was reduced by 40% (p less than 0.01) compared with citrate-saline treated control rats. No significant lymphocytic infiltration was detectable in islets, but the exocrine pancreas exhibited inflammatory lesions. Degranulation and vacuolation was evident by ultrastructural analysis. Following administration of CFA or STZ alone, pancreatic insulin content was insignificantly reduced, and major histopathological changes in pancreatic islets were apparently absent. These results support the hypothesis that activation of the immune system by CFA allows an anti-beta cell reaction to develop following exposure to the beta cell toxic agent STZ.

Serum activities of complement-dependent antibody mediated cytotoxicity (C'AMC) were determined in 36 consecutive patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM). The sequential exposure of 51Cr labeled neonatal rat islet cells to patient serum and rabbit complement revealed the presence of C'AMC in 28 IDDM subjects. The C'AMC titres ranged between 1:4 and 1:512 and were not related to the C'AMC activity of a given sample as measured at a standard dilution (1:4). In comparison to the clinical characteristics of 21 IDDM patients with negative C'AMC, higher C'AMC titres (greater than or equal to 1:32) were associated with a lower mean age at diagnosis of IDDM (12.2 +/- 2.1 vs. 19.0 +/- 2.3 years; p less than 0.05), with a higher frequency of infections up to 6 months prior to diagnosis (6 out of 11 vs. 3 out of 21 patients; p less than 0.05) and, although statistically not significant, a preponderance of female sex together with a decreased frequency of HLA-DR4. In contrast, fasting C-peptides levels, HLA-DR3 antigen frequency and Coxsackie B1-6 virus antibody titres were not related to the C'AMC titres. It is concluded that (1) C'AMC titration is superior to the detection of initial C'AMC levels for evaluating the strength of the complement-dependent humoral immune response towards islet cell surface (auto)antigen(s), and (2) infectious agents may be involved in eliciting a C'AMC response.

A sensitive and versatile radioimmunoassay (RIA) for insulin was established using human insulin standard, a specific guinea pig anti-insulin antiserum and rabbit anti-guinea pig serum. Radioiodination was performed according to a modified chloramine T method. Tracer preparations were used for as long as 6 weeks after iodination. The standard curve ranges from 0.044 to 1.2 nmol/l. The intra-assay coefficient of variation (CV) was 3-5% and the inter-assay CV was 6-9% in the optimal range between 0.4 and 0.9 nmol/l. The average recovery of human insulin added to plasma or serum samples was 100.2 +/- 2.0% (n = 38) and 100.1 +/- 1.9% (n = 42), respectively. In addition to human insulin, porcine, canine, rabbit and bovine insulin can also be determined but not rat or mouse insulin. The cross-reactivity of the antiserum with porcine proinsulin was found to be 40% on the molar basis. The range of mean fasting plasma insulin concentrations in healthy subjects and under various pathological conditions were estimated.